Quartz crystal microbalance and voltammetric measurements are used to study the electroenzymatic activity of incorporated bacterial polyhemic cytochrome c(3) in clay minerals. Two different natural clays have been chosen, i.e., kaolinite and montmorillonite, which differ in structure, swelling property and cation exchange capacity. Comparative voltammetric studies of various electroactive species at clay-modified pyrolytic graphite electrodes are first undertaken, which allow the complete characterization of the clay deposit. Then, the incorporation process of bacterial polyhemic cytochrome c(3) in the two clays is studied coupling quartz crystal microbalance and voltammetric measurements. Based on their respective characteristics, each clay yields a different behavior of the incorporated cytochrome c(3), with significantly different electroactive fractions of the immobilized protein. The consequences of the interactions between the protein and the clays on the enzymatic activity of cytochrome c(3) is then further examined. Firstly, the influence of the incorporation of cytochromes in clays on the metal reductase activity developed by bacterial polyheme c-type cytochromes is discussed. Secondly, hydrogenase enzymatic reactions are studied inside the clay films. An electrode is constructed by immobilizing hydrogenase in the clay and progressively incorporating either artificial (methyl viologen) or physiological (cytochrome c(3)) hydrogenase partner. It is shown that this modified electrode yields an efficient and stable response for either H-2 consumption or evolution over a large range of pH. (c) 2005 Elsevier B.V. All rights reserved.